Project Summary Diabetic retinopathy (DR) is a leading cause of vision loss and impairment worldwide. Diabetic macular edema (DME) as a result of blood-retinal barrier (BRB) breakdown is a major complication of DR leading to blindness. Despite the success of anti-vascular endothelial growth factor (VEGF) therapy on DME, little is known about placental growth factor (PlGF)'s functional role in BRB breakdown in DR. PlGF, a member of the VEGF sub- family, is a multifunctional cytokine with pathological angiogenic properties. Recent studies highlight PlGF's role in BRB breakdown in DME. We recently showed that PlGF knockout (KO) mice were protected from diabetes- caused BRB breakdown by upregulating several protective proteins. Emerging clinical studies showed that the drug aflibercept, which blocks both PlGF and VEGF, prevented BRB breakdown in DME patients. Despite these recent advances several questions remain to be addressed. 1) Is selective PlGF inhibition sufficient to prevent diabetes-caused BRB breakdown? 2) What is the cell type-specific effect on BRB function by targeting PlGF? 3) Does PlGF regulate BRB by mechanisms distinct from VEGF? 4) Are there interactions between PlGF and VEGF (PlGF-VEGF heterodimers) that together contribute to BRB breakdown in DR? 5) What is the role of VEGF receptor (VEGFR1) signaling in the regulation of human retinal endothelial cell (HREC) barrier function? The answers to these important questions will better define PlGF's causal role in diabetes-induced BRB breakdown that will lead to the design of better precision-targeted treatments for DME patients. Therefore, the objective of this proposal is to address these questions. Our overall hypothesis is that targeting PlGF prevents diabetes-caused BRB breakdown via upregulation of protective proteins, disruption of PlGF-VEGF dimers, and inactivation of VEGFR1 in pericyte. Three specific aims are proposed to test the hypothesis. Aim-1 is to determine the role of key survival or antioxidant proteins in BRB protection by targeting PlGF in DR. Small hairpin (sh) RNA and monoclonal antibody will silence or block PlGF in vivo or in vitro. The BRB protection by survival or antioxidant proteins will be elucidated with HREC culture. Aim-2 is to determine the contribution of PlGF-VEGF heterodimers on BRB breakdown in DR. PlGF KO mice will be used to determine if PlGF is essential for DR-like features by VEGF. Insulin will treat diabetic mice to determine if hypoglycemia induces PlGF-VEGF dimerization. A dominant-negative PlGF variant, which can heterodimerize with VEGF but not bind VEGFR1, will determine the role of PlGF-VEGF in diabetes-induced BRB breakdown. Aim-3 is to determine the role of VEGFR1 signaling in pericyte and its role in paracrine regulation of BRB function. The proposed paracrine mechanism(s) will be deciphered: pericyte VEGFR1 signaling cascades triggered by high glucose (HG) leads to upregulation of VEGF, PlGF, and/or VEGF-B expression and induction of VEGFR1 phosphorylation or activation events that mediate HG-induced pericyte apoptosis (via nuclear factor (NF)-?B). The damaged pericytes contribute to retinal EC barrier dysfunction by disrupting the balance between Angiopoietin (Ang)-1 and Ang-2.